ES2198488T3 - PROTECTORS - Google Patents

Abstract

Peptide-based compounds containing four invariant cycteine residues which have been optionally oxidized to contain two intramolecular disulfide bonds, or modified forms where the cysteines are replaced are useful as preservatives and in preventing, treating, or ameliorating viral or microbial infection in animals and plants, and in inactivating endotoxin. These compounds, in one embodiment, are of the formula: A1A2A3A4A5C6A7C8A9A10 (1) A11A12C13A14C15A16(A17A18) and the N-terminal acylated and/or C-terminal amidated or esterified forms thereof, which is either in the optionally -SH stabilized linear or in a cystine-bridged form wherein each of A1 and A9 is independently a basic amino acid; each of A2 and A3 is independently a small amino acid; each of A5, A7, A12, A14 and A16 is independently a hydrophobic amino acid; A4 is a basic or a small amino acid; A10 is a basic or a small amino acid or is proline; A11 is a basic or a hydrophobic amino acid; A17 is not present or, if present, is a small amino acid; A18, is not present or, if present, is a basic amino acid; or a modified form of formula (1) and the N-terminal acylated and/or C-terminal amidated or esterified forms thereof wherein each of 1-4 cysteines is independently replaced by a hydrophobic amino acid or a small amino gacid.

Description

Protegrins

Technical field

The invention relates to the field of peptides antibiotics In particular, the invention concerns short peptides, some of which are isolated from porcine leukocytes, which They have a wide range of antimicrobial activities.

Prior art

One of the defense mechanisms against infection by both animals and plants is the production of peptides that have antimicrobial and antiviral activity. Various classes of these peptides have been isolated from tissues of both plants and animals. A well known class of such peptides is taquiplesin that was first isolated from horseshoe crab hemocytes as described by Nakamura, T. et al. J Biol Chem (1988) 263 : 16709-16713. This article described the initial isolated taquiplesin, Taquiplesina I, of the Japanese species. Taquiplesin I is a 17 amino acid amidated peptide that contains four cysteine residues that provide two intramolecular cystine bonds. A later article by this group, Miyata, T. et al. J Biochem (1989) 106 : 663-668, documents the isolation of a second taquiplesin, Taquiplesin II, which consists of 17 amidated residues at the C-terminal end, which It also contains four cysteine residues and two intramolecular disulfide bonds. Two additional 18 mer peptides, called polifemusins, highly homologous to Taquiplesin II and containing the same positions for all four cysteine residues, were also isolated from American horseshoe crabs. Polifemusin I and Polifemusin II differ from each other only by replacing an arginine residue with a lysine. All peptides were described as having an antifungal and antibacterial activity. A later article by Murakami, T. et al. Chemotherapy (1991) 37 : 327-334, describes the antiviral activity of taquiplesin with respect to the vesicular stomatitis virus; Herpes simplex I & II, Adenovirus I, Rovirus II and Poliovirus I viruses were resistant to deactivation by Taquiplesina I. Morimoto, M. and other Chemotherapy (1991) 37 : 206-211, found that Taquiplesin I was inhibitory of HIV. This anti-HIV activity was also found to be possessed by a synthetic analogue of Polifemusin II as described by Nakashima, H. and other Antimicrobial agents and chemotherapy (1992) 1249-1255. Antiviral peptides have also been found in rabbit leukocytes as documented by Lehrer, RI and others J Virol (1985) 54 : 467-472.

Other important classes of cysteine-containing antimicrobial peptides include defensins, β-defensins and insect defensins. Defensins are somewhat longer peptides characterized by six invariant cysteines and three intramolecular cystine disulfide bonds. Defensins were described by Lehrer, RI and others Cell (1991) 64 : 229-230; Lehrer, RI et al. Ann Rev Immunol (1993) 11 : 105-128. A review of mammalian derived defensins by Lehrer, RI and others is found in Annual Review Immunol (1993) 11 : 105-128; three patents have been published on the defenses: US patent documents US 4,705,777; US 4,659,692; and US 4,543,252. Defensins have been found in polymorphonuclear neutrophils (PMN) of humans and several other animals, as well as in rabbit pulmonary alveolar macrophages, and in epithelial cells (Paneth) of the murine small intestine and in corresponding cells in humans.

Β-defensins are found in bovine respiratory epithelium cells, in bovine granulocytes and in bird leukocytes. See Selsted NE and others . J Biol Chem (1993) 288 : 6641-6648 and Diamond, G. and others Proc Natl Acad Sci (USA) (1989) 88 : 262-265.

Antifungal and antibacterial peptides and proteins have also been found in plants (Broekaert, WF and other Biochemistry (1992) 31 : 4308-4314) as reviewed by Cornelissen, BJC and other Plant Physiol (1993) 101 : 709-712. Expression systems for the production of such peptides have been used to transform plants to protect plants against such infection as described, for example, by Haln, R. and others Nature (1993) 361 : 153-156.

The present invention provides a new class of antimicrobial and antiviral peptides, designated herein as `` protegrins '', representative members of those who have been isolated from porcine leukocytes. These peptides are useful as antibacterial, antiviral and antifungal agents in both plants As in animals.

The isolation of the protegrin peptides of the invention was documented by current applicants in an article by Kokryakov, VN and other FEBS (1993) un337 : 231-236 (July article). A subsequent publication of this group described the presence of a new protegrin, whose sequence, and that of its precursor, was deduced from its isolated cDNA clone. Zhao, C et al. , FEBS Letters (1994) 346 : 285-288. An additional article describing cationic peptides from porcine neutrophils was published by Mirogorodskaya, OA and other FEBS (1993) 330 : 339-342 (September article). Storici, P. et al. Biochem Biophys Res Comm (1993) 196 : 1363-1367, documents the recovery of a DNA sequence encoding a porcine leukocyte antimicrobial peptide with a catechine-like prosequence. It is documented that the peptide is one of the protegrins described here below. Patent document W095 / 03325 describes short protegrin peptides, some of which are isolated and purified from porcine leukocytes having an antimicrobial activity. Additional publications related to protegrins are Harwig, SSL, and other J. Peptide Sci . (1995) in the press; and Zhao, C., and others FEBS-MS MB-283 (1995) in press.

It has also been found that the protegrins of the invention bind endotoxins, that is, lipopolysaccharide (LPS) compositions derived from gram-negative bacteria believed to be responsible for gram-negative sepsis. This type of sepsis is an extremely common state and is often fatal. Others have attempted to design and study the proteins that bind LPS / endotoxins, and documents illustrating these attempts appear in Rustici, A. and others Science (1993) 259 : 361-364; Matsuzaki, K. et al. Biochemistry (1993) 32 : 11704-11710; Hoess, A. and others EMBO J (1993) 12 : 3351-3356; and Elsbach, P. et al. Current Opinion in Immunology (1993) 5 : 103-107. The protegrins of the present invention provide additional compounds that are capable of deactivating LPS and improving its effects.

In addition to the foregoing, the protegrins of the invention are effective in inhibiting the growth of organisms that are associated with sexually transmitted diseases. It has been estimated that 14 million people worldwide are infected with HIV and that millions of women suffer from pelvic inflammation every year. Chlamydia trachomatis and Neisseria gonorrhoeae cause more than half of these inflammatory diseases although E. coli , Mycoplasma hominis and other infectious microorganisms may also be responsible. Pathogens include viral, bacterial, fungal and protozoic pathogens. It is especially important that the antibiotics used to fight these infections be effective in physiological states. The protegrins of the present invention offer these properties.

Description of the invention

In one embodiment, the invention is directed to the peptides of 16-18 amino acid residues characterized by four invariant cysteines and both by a characteristic pattern of basic and hydrophobic amino acids and by being insulated from animal leukocytes using the method of the invention. In a second embodiment, the invention is directed to the above peptides in which 1-4 of these cysteines is replaced by a small or hydrophobic amino acid. All of these peptides can be produced synthetically and some can be produced recombinantly or can be isolated from their natural sources and purified for use as preservatives or in pharmaceutical compositions to treat or prevent infections in animals. Alternatively, the peptides can be formulated in compositions that can be applied to plants to protect them against viral or microbial infections. In another additional approach, the DNA encoding the peptides can be expressed in situ , in animals or preferably in plants, to fight infections. Peptides are also useful as standards in antimicrobial assays and for binding endotoxins.

Consequently, in one aspect, the invention is directed to a compound purified and recombinantly isolated or produced from the formula A_ {1} -A_ {2} -A_ {3} -A_ {4} -A_ {5} -C_ {6} -A_ {7} -C_ {7} -A_ {9} -A_ {10} - A_ {11} -A_ {12} - C_ {13} -A_ {14} -C_ {15} -A_ {16} - (A_ {17} -A_ {18}) \ eqnum {(1)} and the acylated and / or C-terminal acylated or esterified N-terminal forms thereof, which are both in the linear form optionally stabilized in the -SH and in the form joined by cystine bridges

wherein A 1 is a basic amino acid;

each of A_ {2} and A_ {3} is independently a small amino acid;

each of A_ {5}, A_ {7}, A_ {14} is independently a hydrophobic amino acid;

A4 is a basic or small amino acid;

each of A_ {9}, A_ {12}, and A_ {16} is independently a basic, hydrophobic, neutral / polar amino acid or small;

A 10 is proline;

A 11 is a basic, neutral / polar amino acid, hydrophobic or small or is proline;

A_ {17} is not present or, if it is, it is a basic, neutral / polar, hydrophobic or small amino acid;

A_ {18} is not present or, if it is, it is a basic, hydrophobic, neutral / polar or small amino acid, or a form modified from Formula (1) and acylated N-terminal and / or amidated C-terminal or esterified forms of same in which at least one of the 4 cysteines is replaced independently by a hydrophobic amino acid or an amino acid small;

Provided that the compound of Formula (1) must have a load of +3 or greater.

In other additional aspects, the invention is directed to recombinant materials useful for the production of the peptides of the invention as well as to plants or animals modified to contain expression systems for the production of these peptides. The invention is also directed to pharmaceutical compositions and compositions for application in plants containing the peptides of the invention as well as active ingredients or compositions containing expression systems for the production of the peptides or for in situ expression of the nucleotide sequence coding for these peptides. The invention is also directed to methods for preparing the peptides of the invention synthetically, to antibodies specific for these peptides, and for the use of the peptides as preservatives.

In other aspects, the invention is directed to use of the compounds of the invention as standards in tests antimicrobials The compounds can also be used as antimicrobials in solutions useful for eye care, such as contact lens solutions, and in compositions topical or other pharmaceuticals for the treatment of diseases of sexual transmission (STD). The invention is also directed to the use of the compounds of the invention as preservatives for food or other perishables. How the peptides of the invention can disable endotoxins, the invention is also directed to a method to deactivate endotoxins using the compounds of the invention and to treat gram-negative sepsis by taking advantage of this property.

Brief description of the drawings

Figure 1 shows the elution pattern of a ultrafiltrate concentrate of porcine leukocytes applied to a Biogel column P10.

Figure 2 shows the antibacterial activity of the P10 fractions obtained from the elution of the described column in Figure 1.

Figure 3 shows an elution pattern obtained when fractions 76-78 of the Biogel column P10 of Figure 1 apply to HPLC.

Figure 4 shows the antimicrobial activity of the purified porcine protegrins of the invention:

Figure 4a shows antibacterial activity against E. Coli ;

Figure 4b shows antibacterial activity against Listeria monocytogenes ;

Figure 4c shows antifungal activity against Candida albicans ;

Figure 4d shows antibacterial activity against S. aureus .

Figure 4e shows antibacterial activity against K. pneumoneae ;

Figure 5 shows the effect of various Test conditions of antimicrobial activity:

Figure 5a shows activity against Candida albicans in 100 µM NaCl;

Figure 5b shows activity against E. Coli in 100 µM NaCl;

Figure 5c shows activity against Candida albicans in 90% fetal calf serum.

Figure 6 shows the antimicrobial activity of the linear forms of protegrins in various conditions of test:

Figure 6a shows the activity against E. coli in 10 mM phosphate citrate buffer, pH 6.5;

Figure 6b shows the activity against E. coli in the same buffer with 100 mM NaCl;

Figure 6c shows the activity against L. monocytogenes in the buffer of Figures 6a-6b;

Figure 6d shows the activity against L. monocytogenes in the same buffer with the addition of 100 mM NaCl;

Figure 6e shows the activity against C. albicans in the presence of 10 mM phosphate; Y

Figure 6f shows the activity against C. albicans in the presence of 10 mM phosphate plus 100 mM NaCl.

Figure 7 shows a composite material of cDNA which codes for PG-1 precursors, PG-2, PG-3 and PG-4

Figure 8 shows the nucleotide sequence and the deduced amino acid sequence of the genomic DNA encoding for the precursor protein for the antimicrobial compounds of invention PG-1, PG-3, and PG-5

Figure 9 shows the organization of DNA genome of protegrin.

Figure 10 shows the amino acid sequences of PG-1 to PG-5 protegrins.

Figures 11a-11c show the antimicrobial activity of PG-5 prepared synthetically compared to the prepared PG-1 synthetically

Figures 12a-12d show the effects of various protegrins against various microbes Diana.

Figure 13 shows a graphic representation of the effects of kite and bullet forms of PG-1 against gram-positive bacteria.

Figure 14 shows a graphic representation of the effects of kite and bullet forms of PG-1 against gram-negative bacteria.

Figure 15 is a graphic representation of the antimicrobial activity of the snake form of the PG-1 against gram-positive bacteria.

Figure 16 is a graphic representation of the antimicrobial activity of the snake form of the PG-1 against gram-negative bacteria.

Ways of carrying out the invention

The peptides of the invention are described by the formula: A_ {1} -A_ {2} -A_ {3} -A_ {4} -A_ {5} -C_ {6} -A_ {7} -C_ {8} -A_ {9} -A_ {10} - A_ {11} -A_ {12} - C_ {13} -A_ {14} -C_ {15} -A_ {16} - (A_ {17} -A_ {18}) \ eqnum {(1)} and its modified forms defined. Those peptides that are naturally present must be in purified and isolated form or recombinantly prepared.

The designation A_ {n} in each case represents a amino acid at the position specified in the peptide. As A_ {17} and A18 may or may not be present, the peptides of the invention They contain either 16, 17 or 18 amino acids. The positions of the cysteine residues, shown as C in Formula (1), are invariants in the peptides of the invention; however, in the forms modified peptides of Formula (1), also included within the scope of the invention, at least one of 1-4 of these cysteines can be replaced by an amino acid hydrophobic or small.

The amino terminal of the peptide may be in free amino form or may be acylated by a group of the formula RCO-, in which R represents a hydrocarbyl group of 1-6C. The hydrocarbyl group is saturated or unsaturated and is typically, for example, methyl, ethyl, i-propyl, t-butyl, n-pentyl, cyclohexyl, cyclohexene-2-yl, hexene-3-yl, hexin-4-yl, and the like.

The C-terminal end of peptides of the invention may be in the form of a carboxyl group without derivatize, either as the free acid or as an acceptable salt, such as potassium, sodium, calcium, magnesium, or other salt of an ion inorganic or an organic ion such as caffeine. Carboxyl terminal can also be derivatized by forming a ester with an alcohol of the formula ROH, or it may be amidated by an amine of the formula NH3, or RNH2, or R2 NH, in which each R is independently hydrocarbyl of 1-6C as defined above. Are preferred the amidated forms of the peptides in which the C-terminal has the formula CONH2.

How the peptides of the invention contain numbers substantial basic amino acids, the peptides of the invention can be supplied in the form of acid addition salts. Salts Typical acid additions include those of inorganic ions such as chloride, bromide, iodide, fluoride or the like, sulfate, nitrate, or phosphate, or they can be salts of organic anions such as acetate, formate, benzoate and the like. The acceptability of each of such salts is dependent on the intended use, as commonly understood.

The peptides of the invention containing at least two cysteines may be in the form of a linear or cyclic chain. Linear chain forms are convertible to cyclic forms, and vice versa . Methods for forming disulfide bridges to create cyclic peptides are well known in the art, since they are methods for reducing disulfides to form linear compounds. Linear compounds can be stabilized by the addition of a suitable alkylating agent such as iodoacetamide.

Cyclic forms are the result of cystine junction formation between all or some of the four invariant cysteine residues. The cyclic forms of the invention include all possible permutations of link formation cystine; if the cysteines are numbered in order of appearance starting at the N-terminal as C 6, C 8, C_ {13} and C_ {15}, these permutations include:

C 6 -C 8;

C 6 -C 13;

C 6 -C 15;

C 8 -C 13;

C 8 -C 15;

C 13 -C 15;

C 6 -C 8, C 13 -C 15;

C 6 -C 13, C 8 -C 15; Y

C 6 - C 15, C 8 - C 13.

In the modified forms of the peptides, in the that cysteines 1-4 are replaced, similar permutations are available when 2-3 cysteines are present.

The native forms of protegrins contain two cystine bonds that are between the cysteines in the position 6 and the cysteine at position 15 and the other between the cysteines in position 8 and cysteine in position 13. Consequently, in those embodiments that have two cystine bonds, the form C_ {6} -C_ {15}, C_ {8} -C_ {13} It is preferred. However, it has been discovered by current applicants that the forms of protegrins that contain only A cystine junction are active and easily prepared. They are preferred among embodiments that have a single junction of cystine those represented by C 6 -C 15 alone and by C_ {8} -C_ {13} alone.

The forms that contain a cystine C 6 -C 15 as the only cystine junction is they generally designate as `` bullet '' forms of protegrins; those in which the only cystine is C_ {8} -C_ {13} are designated as `` comet '' forms. Kite and bullet shapes can be very conveniently made by replacing the cystines in positions that do not are bound by cystine with a neutral amino acid, preferably a small amino acid such as glycine, serine, alanine or threonine and less preferably by a polar neutral amino acid such as asparagine or glutamine. Thus, in embodiments of the bullet form, each of C 8 and C 13 is independently alanine, serine, threonine or glycine, preferably both are alanine. By on the contrary, in the form of a comet, C_ {6} and C_ {15} are like this replaced.

As the linearized forms of the peptides native cyclists have valuable activities, even when they chemically stabilize to preserve the sulfhydryl form of cysteine for example, by reaction with iodoacetamide, the Compounds of the invention also include linearized forms that They are stabilized with suitable reagents. As defined here, the `` SH- stabilized '' forms of the peptides of the invention contain sulfhydryl groups reacted with reagents standards to prevent reformation in disulfide bonds.

An alternative approach to provide Linear forms of the protegrins of the invention comprise the use of the modified form of the peptides in which the residues of cysteine are replaced by amino acids that do not form bonds of Cystine In this case, too, the 4 (or at least 3) of the cysteines at positions 6, 8, 13, and 15 are replaced by polar or small neutral amino acids as indicated above. It is preferable that the 4 cysteine residues be replaced for minimize the probability of intermolecular bonds.

The amino acids indicated by A n can be those encoded by the gene or analogs thereof, and may be also the D isomers thereof. A preferred embodiment of the peptides of the invention is that way in which all residues they are in configuration D thereby giving resistance to protease activity while retaining properties antimicrobial or antiviral. The resulting protegrins are them same enantiomers of the forms containing L-amino acids native people.

The notations of the amino acids used here are Conventional and are as follows:

Amino acid One letter symbol Three symbol letters To the girl TO To Arginine R Arg Asparagine N Asn Acid aspartic D Asp Cysteine C Cys Glutamine Q Gln Acid glutamic AND Glu Glycine G Gly Histidine H His Isoleucine I Ile Leucine L Leu

(Continuation)

Amino acid One letter symbol Three symbol letters Lysine K Lys Methionine M Met Phenylalanine F Phe Proline P Pro Serina S Be Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr valine V val

The non-genetically encoded amino acids are Abbreviate as indicated in the discussion below.

In the specific peptides shown in the present application, the L form of any amino acid residue that having an optical isomer is desirable unless form D is expressly indicated by a dagger symbol (^ \ dagger).

The compounds of the invention are peptides that are partially defined in terms of amino acid residues of designated classes. The amino acid residues may be generally subclassified into major subclasses like follow:

Acids: The residue has a negative charge due to the loss of the H ion at a physiological pH and the residue is attracted by an aqueous solution so that it looks for positions on the surface in the conformation of a peptide in which it is content when the peptide is in an aqueous medium at a pH physiological.

Basic: The residue has a positive charge due to the association with H ion at a physiological pH and the residue is attracted by an aqueous solution so that it looks for positions on the surface in the conformation of a peptide in which it is content when the peptide is in an aqueous medium at a pH physiological.

Hydrophobic: the residues are not charged at a pH physiological and the residue is repelled by an aqueous solution of so that it looks for the internal positions in the conformation of a peptide in which it is contained when the peptide is in a medium aqueous.

Neutral / polar: Waste is not charged to a physiological pH, but the residue is not sufficiently repelled by aqueous solutions so as to look for the internal positions in the conformation of a peptide in which it is contained when the Peptide is in an aqueous medium.

This description also characterizes certain amino acids as `` small '' since their side chains do not they are long enough, even if polar groups are missing, to confer hydrophobicity. `` Small '' amino acids are those with four carbons or less when at least one polar group is in the side chain and three carbons or less when not.

It is understood, of course, that in a collection individual waste molecule statistics some molecules will be charged, and others will not, and that there will be an attraction for or a repulsion from an aqueous medium to a greater or lesser extent. For adapt the definition of `` loaded '', a significant percentage (at least about 25%) of the individual molecules They are charged at a physiological pH. The degree of attraction or repulsion required for classification as polar or non-polar is arbitrary and therefore specifically contemplated amino acids by the invention they have been classified as one or the other. most of amino acids not specifically named may be classified based on known behavior.

The amino acid residues may also be subclassified as cyclic or non-cyclic, and aromatic or not aromatic, self-explanatory classifications with respect to substituent groups of the side chain of the residues, and as Small or big The residue is considered small if it contains a total of four carbon atoms or less, including carbon from carboxyl, as long as an additional polar substituent is Present; but three or less. Small waste is always, for Of course, not aromatic.

For the protein amino acids present from naturally, subclassifications according to the previous scheme are as follows.

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Acid : aspartic acid and glutamic acid;

Basic : non-cyclic: Arginine, Lysine; Cyclic: Histidine;

Small : Wisteria, Serine, Alanine, Threonine;

Polar / large : Asparagine, Glutamine;

Hydrophobic : Tyrosine, Valine, Isoleucine, Leucine, Methionine, Phenylalanine, Tryptophan.

The secondary amino acid proline encoded by a gene is a special case due to its known effects in the secondary conformation of the peptide chains, and therefore, It is not included in a group. Cysteine residues are not either included in these classifications since their ability to form disulfide bonds to provide a secondary structure It is critical in the compounds of the present invention.

Certain commonly found amino acids, which are not encoded by the genetic code, include, for example, beta-alanine (beta-Ala), or others omega amino acids, such as 3-aminopropionic, 2,3-diaminopropionic (2,3-diaP), 4-aminobutyric acid and so on, acid alpha-aminisobutyric (Aib), sarcosine (Sar), Ornithine (Orn), citrulline (Cit), t-butylalanine (t-BuA), t-butyl glycine (t-BuG), N-methylisoleucine (N-MeIle), phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine (Nle), 2-naphthylalanine (2-Nal); acid 1, 2, 3, 4-tetrahydro-isoquinolin-3-carboxylic (Tic); β-2-thienylalanine (Thi); methion sulfoxide (MSO); and homoarginine (Har). These too They fall into conveniently particular categories.

Based on the previous definitions,

Sar, beta-Ala, 2, 3-diaP and Aib are small;

t-BuA, t-BuG, N-MeIle, Nle, Mvl, Cha, Phg, Nal, Thi and Tic are hydrophobes;

Orn and Har are basic;

Cit, Acetil Lis, and MSO are neutral / polar.

The various omega amino acids are classified according to size as small (beta-Ala and 3-aminopropionic) or as large and hydrophobic (everyone else).

Other amino acid substitutions of those amino acids encoded in the gene may also be included in peptide compounds within the scope of the invention and may be classified within this general scheme according to their structures

In all the peptides of the invention, one or more amide linkages (-CO-NH-) may be optionally replaced with another linkage that is an isoster such as -CH2 NH-, -CH2S-, -CH_ {2CH2, -CH = CH- (cis and trans), -COCH2-, -CH (OH) CH2- and -CH2SO-. This replacement can be performed by methods known in the art. The following references describe the preparation of analogous peptides that include these alternative binding moieties: Spatola, AF, Vega Data (March 1983), Vol. 1, number 3, `` Peptide Backbone Modifications '' (general review); Spatola, AF, in `` Chemistry and Biochemistry of Amino Acids Peptides and Proteins, '' B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983) (general review); Morley, JS, Trends Pharm Sci (1980) pp. 463-468 (general review); Hudson, D., et al., Int J Pept Prot Res (1979) 14 : 177-185 (-CH 2 NH -, - CH 2 CH 2 -); Spatola, AF, and others, Life Sci (1986) 38 : 1243-1249 (-CH_ {2} -S); Hann, MM, J Chem Soc Perkin Trans I (1982) 307-314 (-CH-CH-, cis and trans); Almquist, RG, and others, J Med Chem (1980) 23 : 1392-1398 (-COCH2 -); Jennings-White, C., and others, Tetrahedron Lett (1982) 23 : 2533 (-COCH 2 -); Szelke, M., et al., European Application EP 45665 (1982) CA: 97 : 39405 (1982) (-CH (OH) CH 2 -); Holladay, MW, and others Tetrahedron Lett (1983) 24 : 4401-4404 (-C (OH) CH 2 -); and Hruby, VJ, Life Sci (1982) 31 : 189-199 (-CH_ {2} -S-).

The compounds of Formula (1) are generally defined as A_ {1} -A_ {2} -A_ {3} -A_ {4} -A_ {5} -C_ {6} -A_ {7} -C_ {7} -A_ {9} -A_ {10} - A_ {11} -A_ {12} - C_ {13} -A_ {14} -C_ {15} -A_ {16} - (A_ {17} -A_ {18}) \ eqnum {(1)} and the acylated and / or C-terminal acylated or esterified N-terminal forms thereof, which are both in the linear form optionally stabilized in the -SH and in a cystine bridge-bound form,

wherein A 1 is a basic amino acid;

each of A_ {2} and A_ {3} is independently a small amino acid;

each of A_ {5}, A_ {7}, A_ {14} is independently a hydrophobic amino acid;

A4 is a basic or small amino acid;

each of A_ {9}, A_ {12}, and A_ {16} is independently a basic, hydrophobic, neutral / polar amino acid or small;

A 10 is proline;

A 11 is a basic, neutral / polar amino acid, hydrophobic or small or is proline;

A_ {17} is not present or, if it is, it is a basic, neutral / polar, hydrophobic or small amino acid;

A_ {18} is not present or, if it is, it is a basic, hydrophobic, neutral / polar or small amino acid, or a form Modified Formula (1) and N-terminal forms acylated and / or C-terminal amidated or esterified from same in which at least one of the 4 cysteines is replaced independently by a hydrophobic amino acid or an amino acid small;

provided that the compound of Formula (1) must have a load of +3 or greater.

In the preferred embodiments of the compounds of the invention, each of A 1 and A 9 is independently selected from the group consisting of R, K and Har; plus preferably, that both A 1 and A 9 are R.

In another class of preferred embodiments, each one A_ {2} and A_ {3} is independently selected from the group consisting of G, A, S and T; more preferably, than A2 and A_ {3} be G.

In another group of preferred embodiments, A_ {4} is selected from the group consisting of R, K, Har, G, A, S and T; more preferably, that A 4 is R or G.

In another group of preferred embodiments, each one of A_ {5}, A_ {14} and A_ {16} is independently independently selected from the group consisting of I, V, L, Nle and F; preferably I, V, L and F.

In another group of preferred embodiments, each one of A_ {7} and A_ {12} is independently selected from group consisting of I, V, L, W, Y and F; preferably that A7 be Y and A_ {12} be I or F.

In another group of preferred embodiments, A_ {11} is R or W.

A_ {17}, when present, is preferably G, A, S or T, more preferably G;

A_ {18}, when present, is preferably R, K or Har, more preferably R.

As described above, the compounds of Formula (1) are both cyclic and non-cyclic (linearized) or may be modified in which 1-4 of the cysteines are replaced by a small amino acid residue or a hydrophobic residue or a residue of large non-polar amino acid If the linearized forms of the compound of Formula (1) are prepared, or if the linearized forms of these modified peptides that contain at least two cysteines are prepared, it is preferable that the sulfhydryl groups are stabilized by the addition of a suitable reagent. The realizations preferred for hydrophobic amino acids to replace residues of Cysteine are I, V, L and NLe, preferably I, V or L. preferred small amino acids to replace residues of cysteine include G, A, S and T, more preferably G. The Preferred large polar amino acids are N and Q.

In an alternative embodiment, the peptides of the invention are defined as described by Formula (1), but in which the definitions of A_ {n} in each case are determined by the isolation of the peptide from animal leukocytes by the method of the invention. The method of the invention comprises the steps of providing an ultrafiltrate of a leukocyte lysate of animal and isolate peptides from 16-18 amino acids.

These peptides can also be defined by the ability of the DNA that encodes them to hybridize under conditions rigorous with the DNA encoding the exemplified peptides as PG-1, PG-2, PG-3, PG-4 and PG-5 here present.

The compounds of the invention particularly Preferred are:

Unmodified forms

PG-5: R-G-G-R-L-C-Y-C-R-P-R-F-C-V-C-V-G-R PG-5: R-G-G-R-L-C-Y-C-R-P-R-F-C-V-C-V-G-R IB-324: R-G-G-G-L-C-Y-C-R-P-R-F-C-V-C-V-G-R-OH IB-218: R-G-G-G-L-C-Y-C-F-P-K-F-C-V-C-V-G-R

both linear and mono- and bicyclic forms of same, and including acylated N-terminal forms and C-terminal amidated;

Modified forms

IB-225: R-G-G-G-L-C-Y-A-R-P-R-F-A-V-C-V-G-R IB-226: R-G-G-G-L-C-Y-T-R-P-R-F-T-V-C-V-G-R

both linear and cyclic forms (wherever possible) of it, and including the forms N-terminal acylated and C-terminal amidated

Particularly preferred are the compounds in which a single cystine bond is formed between C6 and C 15 or between C 8 and C 13 in which four compounds that they have a cystine bond between C 8 and C 13 each of C_ {6} and C_ {15} is independently replaced by `` X '' in the that X is a hydrophobic, small, or large polar amino acid. Similarly, in which the single cystine bond is between C 8 and C_ {13}, each of C_ {6} and C_ {15} being replaced independently by X as defined above. Too Preferred are the `` snake '' forms of the compounds of the invention where all 4 cysteines are replaced by X as define above. Particularly Preferred Embodiments of these compounds of the invention include:

Kite Shape-5

28

Bullet Shape-5

29

in which X is as defined previously.

Particularly preferred embodiments of X are those in which X is a small amino acid, especially S and A, especially A.

Preparation of the compounds of the invention

The compounds of the invention, frequently designated here `` protegrins '' are essentially skeletons peptides that may be modified in the N- or C-terminal and they may also contain one or two cystine disulfide bonds. The Peptides can first be synthesized in non-cyclic form. These peptides can then be converted into cyclic peptides if desired by standard methods of cystine bond formation. How applies here to protegrins, the `` cyclic forms '' are refer to those forms that contain cyclic portions under the formation of disulfide bonds between cysteine residues in the peptide If the linear chain shapes are preferred, it is it is preferable to stabilize the sulfhydryl groups for any of the peptides of the invention containing two or more residues of cysteine

Standard methods of synthesis of peptides the size of protegrins. Currently the most commonly used are solid phase synthesis techniques; in actually, you can buy automated equipment for systematic construction of peptide chains. It also can use a liquid phase synthesis but it is considerably less convenient. When synthesized using these standard techniques, the amino acids not encoded by the gene and the D enantiomers can be Use in the synthesis. Thus, a very practical way to obtain the compounds of the invention is to employ these standard techniques of chemical synthesis

In addition to provide the peptide skeleton, the N and / or C-terminal can be derivatized, using again the conventional chemical techniques The compounds of the invention can optionally contain an acyl group, preferably a group acetyl in the amino terminal. Methods to acetylate or, more generally acylate the free amino group in the N-terminal are generally known in the art; in addition, the N-terminal amino acid can be supplied in the synthesis in the acylated form.

In the carboxy terminal end, the group carboxyl can, of course, be present in the form of a salt; in the case of pharmaceutical compositions this will be a salt pharmaceutically acceptable. Suitable salts include those formed with inorganic ions such as NH 4 +, Na +, K +, Mg ++, Ca ++, and the like as well as salts formed with organic cations such as those of caffeine and other highly substituted amines. The carboxy terminal end it can also be esterified using alcohols of the formula ROH in which R is hydrocarbyl (1-6C) as defined previously. Likewise, the carboxy terminal end may be amidated to have the formula -CONH2, CONHR, or -CONR2, in which each R is independently hydrocarbyl (1-6C) as defined here. Techniques for esterification and amidation as well as neutralization in the presence of the base to form salts are all standard chemistry techniques organic

If the peptides of the invention are prepared in physiological conditions, the amino groups of the side chain of the basic amino acids will be in the form of addition salts Acid relevant.

The formation of disulfide bonds, if desired, is carried out in the presence of mild oxidizing agents. Oxidizing chemical agents may be used, or the compounds may simply be exposed to the oxygen in the air to effect these bonds. Various methods are known in the art. Useful processes for the formation of disulfide bonds have been described by Tam, JP et al., Synthesis (1979) 955-957; Stewart, JM et al., `` Solid Phase Peptide Synthesis '' 2d Ed. Pierce Chemical Company Rockford, IL (1984); Ahmed AK et al., J Biol Chem (1975) 250 : 8477-8482 and Pennington MW et al., Peptides 1990 , E. Giralt et al., ESCOM Leiden, The Netherlands (1991) 164-166. An additional alternative is described by Kamber, B. et al., Helv Chim Acta (1980) 63 : 899-915. A method carried out on solid supports is described by Albericio Int J Pept Protein Res (1985) 26 : 92-97.

A particularly preferred method is an oxidation solution that uses molecular oxygen. This method has been used by the inventors here to replicate synthetic PG-1, PG-3 in their amide or acidic forms, PG-1 enanthium and the two PG-1 (C 6 -C 15) unisulfide compounds and C_ {8} -C_ {13}). The recoveries are as high as 30%.

If the peptide skeleton is comprised entirely of genetically encoded amino acids, or if any portion of it is thus composed, the peptide or the relevant portion can also be synthesized using recombinant DNA techniques. The DNA encoding the peptides of the invention can be synthesized itself using commercially available equipment; The codon choice can be integrated into the synthesis depending on the nature of the host. Alternatively, although less convenient, the DNA can be obtained, at least initially, by screening a cDNA library prepared from porcine leukocytes using PCR primers or probes based on the sequences of the protegrins described herein. This results in the recovery of the sequence coding for the naturally occurring protegrins of the invention. Obtaining this native sequence is significant for purposes other than the synthesis of protegrins per se ; The availability of naturally occurring sequences provides a useful probe to obtain the corresponding DNA encoding protegrins of other species. Thus, cDNA libraries, for example, of leukocytes derived from other animals can be screened using native DNA, preferably under conditions of high restriction. High restriction is as defined by Maniatis, and other Molecular Cloning: a Laboratory Manual 2 nd Ed, Cold Spring Harbor Laboratory Press (1989), the relevant portions of which are incorporated herein by reference. This procedure also allows recovering allelic variants of these peptides from the same species.

Alternatively, protegrins can be prepare by isolating from leukocytes from a desired species using techniques similar to those described here for the isolation of porcine protegrins. In general, you are techniques involve the preparation of the lysate of a preparation of leukocytes, the ultrafiltrate of the clarified lysate supernatant and recovery of ultrafiltrate. The ultrafiltrate undergoes then to chromatographic separation. The location of the fragments that have anitimicrobial and antiviral activity corresponding to protegrins can be evaluated using criteria of molecular weight and testing the fractions in regards to the desired activities as described here. Native forms of these peptides are believed to be in cyclic form; if desired, linearized forms can be prepared by treating the peptides with reducing agents and stabilizing the sulfhydryl groups that result.

Isolated and produced forms recombinant protegrins may require derivatizations later to modify the N-terminal end and / or the C-terminal end and, depending on the isolation procedure, make the formation of links Cystine as described above. Depending on the organism host used for recombinant production and animal source from which the protein is isolated, some or all of these conversions They may have already been made.

For recombinant production, the DNA that codes for the protegrins of the invention is included in a expression system that places these coding sequences under control of a suitable promoter and other control sequences compatible with a desired host cell. Cell types available hosts cover almost the entire range of the animal kingdom and vegetable Thus, the protegrins of the invention could be produced in bacteria or yeasts (to the point that they can occur in a non-toxic or refractile way or use resistant strains) as well as in animal cells, insect cells and plant cells. Actually, modified plant cells can be used to regenerate plants that contain the relevant expression systems so that the resulting transgenic plant is capable of face-to-face self-protection against these infectious agents.

The protegrins of the invention can be produced in a form that will result in its secretion from the host cell fusing with the DNA that codes for protegrin, a DNA that encodes for a suitable signal peptide, or can be produced intracellularly They can also be produced as proteins from fusion with additional amino acid sequences that may or may not need to be removed later before using these compounds such as antimicrobial or antiviral.

Thus, the protegrins of the invention can be produced in a variety of modalities that include synthesis Chemistry, recombinant production, isolation from sources natural, or some combination of these techniques.

Those members of the protegrin class present from Natural way they are supplied in a purified and isolated way. By `` purified and isolated '' refers free from the environment in which the peptide is normally found (in the case of such peptides naturally present) and in a way that can be used in a practical way. Thus, `` purified and isolated '' form means that the peptide is substantially pure, that is, more than 90% pure, preferably more than 95% pure and more preferably more than 99% pure or in a completely different context such as of a pharmaceutical preparation.

Antibodies

Antibodies against the protegrins of the invention they can also be produced using standard immunological techniques for the production of polyclonal antisera and, if desired, immortalizing host antibody producing cells immunized for monoclonal antibody production sources. Techniques for producing antibodies against Any substance of interest. It may be necessary to enhance the immunogenicity of the substance, particularly as here, in which the material is only a short peptide, coupling the hapten to the vehicle. Suitable vehicles for this purpose include substances that do not produce an immune response in themselves mammal to which the conjugate is administered hapten-vehicle. Common used vehicles include California limpet hemocyanin (KLH), diphtheria toxoid, serum albumin, and the rotavirus protein viral envelope, VP6. The hapten coupling to the vehicle is done by techniques standards such as contacting the vehicle with the peptide in the presence of a dehydrating agent such as the dicyclohexylcarbodiimide or through the use of binding peptides such as those available through the chemical company Pierce Chemical Company, Chicago, IL.

The protegrins of the invention in immunogenic form they are then injected into a suitable mammalian host and the Serum antibody concentrations are monitored. Should note, however, that some forms of protegrins require modification before they are able to cause the antibody production, due to its resistance to processing antigenic For example, the native form of PG-1, which contains two cystine bridges is not immunogenic when it administered without attaching to a larger vehicle and was an immunogen weak even in the presence of potent adjuvants and that when couple to glutaraldehyde or KLH. Applicants believe this is due to its resistance to attack by leukocyte serine proteases (human PMN elastase and cathepsin G) as well as the attack by a aspartic protease (pepsin) that resembles several cathepsins of macrophages The lack of immunogenicity may therefore result from processing resistance towards a linear form that can fit in the pocket that presents the antigen of the cells antigen presenters. The immunogenicity of these forms of the protegrins can be enhanced by cutting the links disulfide

Polyclonal antiserum can be collected when the concentrations are high enough. Alternatively, antibody producing cells of the host such as spleen cells or blood lymphocytes Peripheral can be collected and immortalized. The cells immortalized are then cloned as individual colonies and selected for the production of monoclonal antibodies desired

The antibodies of the invention are, of course, useful in immunoassays to determine the amount or presence of The protegrins. Such tests are essential in the production of controlled quality of the compositions containing the protegrins of the invention. In addition, antibodies can be used to evaluate the efficacy of recombinant production of protegrins, as well as to screen expression libraries in what which concerns the presence of genes that code for protegrins

Compositions containing protegrins and methods of use

The protegrins of the invention are effective in deactivate a wide range of microbial and viral targets, including gram-positive bacteria and Gram-negative yeasts, protozoa and certain strains of virus. Consequently, they can be used in compositions disinfectants and as preservatives for materials such as groceries, cosmetics, medications, or other materials that contain nutrients for organisms. For use in such contexts, protegrins are supplied well as a single protegrin, in mixture with several other protegrins, or in mixture with additional antimicrobial agents. In general, how are you? preservatives in this context, are normally present in relatively small amounts, less than 5%, by weight of the total composition, more preferably less than 1%, even more preferably less than 0.1%.

The peptides of the invention are also useful as standards in antimicrobial assays and in assays to determine the ability of test compounds to bind endotoxins such as lipopolysaccharides.

For use as antimicrobials or antivirals for the treatment of animal subjects, the protegrins of the invention can be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the type of treatment desired - for example, prevention of prophylaxis, therapy; protegrins are formulated in ways consistent with these parameters. A summary of such techniques is found in Pharmaceutical Sciences of Remington, latest edition, Mack Publishing Co., Easton, PA.

Protegrins are particularly attractive as an active ingredient of pharmaceutical compositions useful in the treatment of sexually transmitted diseases, including those caused by Chlamydia trachomatis , Treponema pallidum , Neisseria gonorrhoeae , Trichomonas vaginalis , Herpes simplex type 2 and HIV. Topical formulations are preferred and include creams, ointments, oils, powders, gels and the like. Suitable topical vehicles are well known in the art and can be adapted for particular uses by those professionals of common knowledge.

In general, for use in treatment or STD prophylaxis, the protegrins of the invention can be used alone or in combination with other antibiotics such as erythromycin, tetracycline, macrolides, for example azithromycin and cephalosporins Depending on the mode of administration, the protegrins will be formulated in compositions suitable to allow surface release to affected areas. The protegrins are they can use in forms containing one or two disulfide bridges or They can be linear. In addition, the use of forms enantiomeric containing all D-amino acids they can grant advantages such as resistance to those proteases, such as trypsin and chymotrypsin, for which the protegrins which contain L-amino acids are less resistant.

The protegrins of the invention can be administered individually or as a mixture of several protegrins or in combination with other pharmaceutically active components. Formulations can be prepared in a form suitable for administration systemic or topical or local administration. Formulations Systemic include those designated for injection (for example, intramuscular, intravenous or subcutaneous injection) or it can be prepare for transdermal, transmucosal, or oral administration. The formulation will generally include a diluent as well as, in some cases, adjuvants, buffers, preservatives and the like. The protegrins can also be administered in compositions liposomal or as microemulsions.

If the administration has to be oral, the protegrins of the invention should be protected from degradation in the stomach using a suitable enteric lining. This can avoided to some extent using amino acids in configuration D, thereby providing protease resistance. Without However, the peptide is still susceptible to hydrolysis due to the acidic stomach conditions; that way, it may still be required certain degree of enteric coating.

As described in the examples below, the peptides of the invention retain their activity against microbes in the context of borate solutions that are commonly used in eye care products. It has also been shown that when antimicrobial activity against E. coli has been tested in the presence and absence of lysozyme in buffered borate saline, the presence of lysozyme enhances the effectiveness of PG-3. This effect was more pronounced when PG-3 was autoclaved and similar patterns were obtained for both the acid free form and the amide form. Consequently, protegrins can be used as preservatives in such compositions or as antimicrobials for the treatment of eye infections.

It is particularly important that protegrins retain their activity under physiological conditions including relatively high concentration of saline and in the presence of serum. In addition, protegrins are not cytotoxic with respect to the cells of higher organisms. These properties, described hereinafter in the Examples, make them particularly suitable for in vivo and therapeutic use.

The protegrins of the invention can be applied also to plants or their environment to prevent diseases induced by viruses and microbes in these plants. The compositions suitable for this use will typically contain a diluent as well as a broadcast agent or other subsidiary adjustments beneficial to The plant or the environment.

Thus, the protegrins of the invention can be used in any context in which an antimicrobial and / or antiviral action is required. This use can be a totally in vitro use, or the peptides can be administered to organisms.

In addition, antimicrobial or antiviral activity can be generated in situ by administering an expression system suitable for the production of the protegrins of the invention. Such expression systems can be supplied to plant and animal subjects using known techniques. For example, in animals, expression vectors based on pox viruses can be used to generate the peptides in situ . Likewise, plant cells can be transformed with expression vectors and then regenerated as whole plants that are capable of their own production of the peptides.

A particularly useful property of Protegrins is its activity in the presence of serum. Unlike the defensins, the protegrins are able to exert their effects antimicrobials in the presence of serum.

As shown here below, the protegrins are able to deactivate endotoxins derived from gram-negative bacteria - that is, lipopolysaccharides (LPS) - in standard tests. Consequently, the protegrins can be used in any circumstance in which the LPS deactivation is desired. One such situation is in the treatment or improvement of gram-negative sepsis.

The protegrins of the invention, therefore, they represent a peculiarly useful class of compounds due to the following properties:

1) They have an antimicrobial effect with respect to a broad spectrum of target microbial systems, including viruses, including retroviruses, bacteria, fungi, yeasts and protozoa.

2) Its antimicrobial activity is effective in physiological conditions - that is, physiological saline and in presence of serum.

3) They are not toxic to the cells of organisms superior.

4) They can be prepared in a non-immunogenic way thus extending the number of species to which you can manage.

5) They can be prepared in ways that are resistant to certain proteases that suggest they are antimicrobial even in lysosomes.

6) They can be prepared in ways that resist degradation when autoclaved, thus simplifying its preparation as components of drugs.

The following examples are intended to illustrate but Do not limit the invention.

Example 1 PG-1, PG-2 and insulation PG-3

Fresh swine blood was collected in containers of 15 liters containing 5% EDTA in normal saline, pH 7.4 as an anticoagulant (33 ml / liter of blood). The cells were allowed of the blood will settle for 90 minutes at room temperature and the leukocyte-rich supernatant was removed and centrifuged at 200 x g for 5.7 minutes. The precipitates were gathered and 0.84% resuspended in ammonium chloride to lyse erythrocytes and the resulting leukocytes (70-75% of PMN, 5-10% of eosinophils, 15-25% of lymphocytes and monocytes) were washed in normal saline, resuspended in 10% acetic acid cooled on ice at 10 8 / ml, homogenized and stirred overnight at 4 ° C. The Preparation was centrifuged at 25,000 x g for 3 hours at 4 ° C and the Supernatant was lyophilized and heavy.

950 mg (dry weight) of lyophilized extract, which contained 520 mg of protein by BCA analysis, were stirred overnight at 4 ° C in 100 ml of 10% acetic acid and then centrifuged at 25,000 x g for 2 hours. The supernatant was removed and under pressure through a cell 50 ml ultracentrifugation by shaking (Amicon, Danvers, Ma) that It contained a YM-5 filter. The ultrafiltrate (24.5 mg protein by BCA) was concentrated to 3 ml by centrifugation vacuum (SpeedVac concentrator, Savant Instruments, Hicksville, NY), applied to a column of BioGel P10 2.5 x 117 cm (Bio-Rad, Hercules, CA) and eluted at 4 ° C with acid 5% acetic

Fractions containing 6.6 ml were obtained. The fractions were tested by absorption at 280 nm and the Elution pattern is shown in Figure 1.

Aliquots (66 µl) of each fraction were dried by vacuum centrifugation and resuspended in 6.6 µl of 0.01% acetic acid. Samples of five µl of this concentrate were tested for antimicrobial activity against E. coli ML-35, L. monocytogenes , strain EGD and C. albicans , strain 820, using broadcasting and gel overlapping techniques such as It is described by Lehrer, RI and others J Immunol Meth (1991) 137 : 167-173. Briefly, the coating agars used for all organisms had a final pH of 6.5 and contained 9 mM sodium phosphate / 1 mM sodium citrate buffer, 1% w / v agarose and 0.30 µg / triptocase soy broth powder ml (BBL Cockeysville, MD). The activity units in the radial diffusion test were measured as described; 10 units corresponding to a clear area of 1 mm in diameter around the sample well. The activities obtained for the various fractions are shown in Figure 2. Activity was found in a large number of fractions.

The active fractions were examined additionally by urea acid PAGE (AU-PAGE) and SDS PAGE. The results of these analyzes showed that active antimicrobial peptides of the appropriate molecular weight were present and concentrated in the fractions 76-78.

Fractions 76-78 of the Biogel column P10 were then collected and chromatographed on a 1 x 25 cm Vydac 218 TP1010 column with a gradient (buffer A is 0.1% TFA; buffer B is 0.1% TFA in acetonitrile) the increase in Acetonitrile concentration was 1% per minute. The results, evaluated in terms of absorbance at 280 nm and at 225 nm are shown in Figure 3. The peaks corresponding to the three peptides illustrated here are labeled in the figure. The figure contains also a box that shows the results of a gel PAGE-urea acid stained with Coomassie Blue which It contains an initial mixture composed of assembled fractions and individual PG species. These are labeled M, 1, 2 and 3 in the box. The results clearly show the presence of three different proteins

The isolated proteins were subjected to amino acid analysis using three independent methods, and at Edman degradation, chymotrypsin digestion, and analysis of mass spectrometry by rapid atom bombardment. The peptides, called `` protegrins '', are shown to have the amino acid sequences as follows:

PG-1: RGGRLCYCRRRFCVCVGR

PG-2: RGGRLCYCRRRFCICV

PG-3: RGGGLCYCRRRFCVCVGR,

and are amidated at the end C-terminal

The amidation status of isolated peptides was established by synthesis of PG-3, both in free and carboxyamidated carboxyl forms. These peptides synthetics were then compared to isolate PG-3 using AU-PAGE and also using phase HPLC inverse In both cases, the native product comigrated with the form synthetic amidate

The situation of disulfide bonds in isolated protegrins was also studied using PG-2 as a model. The determination was made using sequential enzyme digestion (chymotrypsin followed by thermolysin) with direct analysis using LC-ESI-MS in the fragments obtained. The results of these analyzes showed that the two intramolecular disulfide bonds were C_ {6} -C_ {15} and C_ {-} {C13}. With the situation of disulfides in these positions, it is likely that the protegrin molecules exist as β sheets taquiplesin-like antiparallel in conformation global.

The above antimicrobial proteins are present in much lower concentrations in initial extracts that rabbit defenses are in raw extracts corresponding in which the defenses constitute more than 15% of the total protein in rabbit granulocytes. Using the method AU-PAGE analytical in various stages of purification, the peptides are only weakly visible in extracts gross while the corresponding gross extracts of rabbit granulocytes clearly show the presence of Defensins The peptides of the invention clearly become evident only after the ultrafiltration stage.

Because the protegrins, whose structures are set out above, show sequence homology in the decapeptide region corresponding to residues 1-10 of the NP-3a rabbit defensin in the decapeptide region at positions 4-13 of PG-3, Protegrins, and in particular PG-3, can share the property of NP-3a defensin in being able to competitively antagonize ACTH-mediated steroid synthesis by adrenocytes. This property, called `` corticostasis, '' can influence the efficacy of protegrins as anti-infective agents when used in vivo .

Example 2 Antimicrobial Activity

The radial diffusion test on agarose gels described in Example 1 was also used to test the activity of purified protegrins. Figures 4a, 4b and 4c show the results against three test organisms in units as described above. Rabbit Defensin (NP-1) and human defensin (HNP-1) They were used as witnesses.

Figure 4a shows that PG-1 and PG-3 are more effective against E. coli ML-35P than HNP-1 and only slightly less effective than NP-1. PG-1 and PH-3 were also effective against Listeria monocytogenes , strain EGD as shown in Figure 4b. In Figure 4c, PG-1 and PG-3 were also effective against Candida albicans . In general, these peptides are about as effective as rabbit NP-1 defensin based on weight and are more effective than HNP-1. In all cases, PG-2 was also effective against the three organisms tested but was not as active as the other two peptides.

In addition to its activity in inhibiting the growth of the aforementioned organisms, PG-1 of the invention has been directly shown to inhibit the growth of Staphylococcus aureus (see Figure 4d) and K. pneumoneae 270 (Figure 4e). The HNP-1 used as a control was less effective against S. aureus and almost entirely ineffective against K. pneumoneae .

The protegrins of the invention have also been tested against various other organisms and show broad spectrum activity. In addition to their efficacy in inhibiting the growth of or infection by microorganisms associated with STDs as described in Example 9 hereinafter, protegrins show strong activity against the following microorganisms in addition to those tested hereinabove: Pseudomonas aeruginosa, Klebsiella pneumoniae , Salmonella typhimurium, Staphylococcus aureus, Histoplasma capsulatum, Myobacterium avium-intracellulare , and Mycobacterium tuberculosis . The protegrins showed only weak activity against Vibrio vulnificus and were inactive against Vibrio cholerae and Borrelia burgdorferi .

Example 3 Conservation of the activity under certain conditions

The antimicrobial activity of the compounds of the invention was tested as set forth above, but under conditions of 100 µM NaCl and in the presence of 90% fetal calf serum. Figures 5a and 5b show that PG-1 and PG-3 retain their activity with respect to C. albicans and E. coli respectively, even in the presence of 100 mM NaCl. Neither NP-1 nor NHP-2 have this property. Figure 5c shows that although NP-1 and NHP-2 lose their ability to deactivate C. albicans in 90% fetal calf serum, deactivation by PG-3 remains.

Consequently, the protegrins of the invention they retain their antimicrobial properties in conditions useful physiological, including isotonic and borate solutions appropriate for use in eye care products.

In addition, synthetic PG-1 was tested for its activity against E. coli ML-35 (serum sensitive) in coating gels containing only 10 mM sodium phosphate buffer, pH 7.4 and a dilution of 1: 100 triptocasa soy broth, both in the presence and absence of 2.5% of normal human serum, which is below the lytic concentration for this strain of E. coli . In the presence of serum, the minimum bactericidal concentration was reduced from approximately 1.0 µg / ml to about 0.1 µg / ml. This type of effect was not observed either by a linear fragment of cathepsin G or by the HNP-1 defensin.

Likewise, using C. albicans as a target organism, coating gels were prepared with 10 mM sodium phosphate with and without 10% normal human serum. The minimum fungicidal concentration dropped from about 1.3 µg / ml in the absence of serum to 0.14 µg / ml in its presence. The serum itself at this concentration did not affect C. albicans .

Thus, the action of the protegrins is not only not inhibited by the presence of the serum but is enhanced in this way. Similar results were obtained using L. monocytogenes as the target organism.

The PG-1 and PG-3 protegrins were incubated for 4 hours at pH 2.0 with 0.5 µg / ml pepsin and then neutralized. The residual antimicrobial activity against C. albicans, E. coli and L. monocytogenes was evaluated and found completely conserved. Similar experiments show that these compounds are not degraded by human leukocyte elastase or by cathepsin G from human leukocytes, even when exposed to high concentrations of these enzymes and at a pH of 7.0-8.0 favorable for proteolytic activity. In addition, synthetic PG-3 amide and synthetic PG-3 acid were autoclaved and tested for antimicrobial activity against E. coli, L monocyogenese and C. albicans ; keeping a total antimicrobial activity in all cases. It is possible that the stability of these compounds to protease degradation and autoclaving is enhanced by the presence of disulfide bonds.

Example 4 Ability to bind endotoxins

The protegrins of the invention were tested for their ability to bind the polysaccharide lipid (LPS) of the gram-negative bacteria of strain 0.55B5 of E. coli . The assay was the Limulus amebocyte lysate (LAL) assay for endotoxins carried out in the presence and absence of the test compounds. The test was carried out using the procedure described in the Sigma Technical Bulletin No. 210 as reviewed in December 1992 and published by Sigma Chemical Company, St Louis, Mo.

The LAL assay is based on the ability of the LPS to affect gelation in the commercial E-Toxate ™ reagent that is prepared from the lysate of circulating amebocytes from the Limulus polyphemus horseshoe crab. As described in the technical bulletin, when exposed to minimum amounts of LPS, the lysate increases in opacity as well as in viscosity and can gel depending on the concentration of endotoxin. The technical bulletin continues to speculate that the mechanism seems analogous to the coagulation of mammalian blood and involves the activation steps of a trypsin-like precoagulation enzyme by LPS in the presence of calcium ion, followed by enzymatic modifications of a `` coagulogen '' by proteolysis to produce a coagulable protein. These stages are considered linked to the biologically active or `` pyrogenic '' portion of the molecule. It has been previously shown that detoxified LPS (or endotoxin) gives a negative LAL assay.

The test compounds were used to various concentrations from 0.25 \ mug-10 \ mug in a final volume of 0.2 ml and the test mixtures contained LPS at a final concentration of 0.05 endotoxin units / ml and E-Toxate ™ at the same concentration. The compounds test were incubated together with the LPS for 15 minutes before that the E-Toxate ™ was added to a final volume after the addition of 0.2 ml of E-Toxate ™. The tubes were then incubated for 30 minutes at 37 ° C and examined in regards to the formation of a gel.

Both isolated native protegrins (nPGs) and synthetically prepared protegrins (sPGs) were tested. The sPGs were prepared with a carboxyl group at the end C-terminal or with one end C-terminal amidated. The nPGs are amidated in the C-terminal end. Six were also tested different rabbit defensins (NPs) and four native defensins of human (HNPs). The results are shown in Table 1.

TABLE 1

Peptide 10 \ mug 5 \ mug 2.5 \ mug 1.0 \ mug 0.5 \ mug 0.25 \ mug nPG-1 without gel without gel without gel without gel + ++ nPG-2 without gel without gel without gel without gel + ++ nPG-3 without gel without gel traces ++ ++ ++ acid sPG-3 without gel without gel traces ++ ++ ++ sPG-3 amide without gel without gel without gel + ++ ++ NP-1 not rehearsed do not rehearsed ++ ++ ++ ++ NP-2 traces? + + ++ ++ ++ NP-3a without gel without gel without gel ++ ++ ++ NP-3b without gel without gel + ++ ++ ++ NP-4 not rehearsed do not rehearsed + ++ ++ ++ NP-5 without gel traces + + ++ ++ HNP-1 without gel + + ++ ++ ++ HNP-2 traces traces traces + + ++ HNP-3 without gel + + ++ ++ ++ HNP-4 without gel traces traces ++ + ++

As seen from the results, all the protegrins, both synthetic and native, and in both amidated forms as non-amidated are capable of joining sufficiently to the LPS to prevent any substantial training gel at concentrations as low as 2.5 µg / 0.2 ml. nPG-1 and nPG-2 are effective at somewhat lower concentrations. The protegrins were substantially more effective than NP or HNP test compounds; the most effective among these witnesses was the NP-3a, a peptide whose primary sequence resembles that of the protegrins

In a follow-up experiment, the LPS concentration varied from 0.05-0.25 endotoxin units (U.E.) and amide PG-3 Synthetic was used as test compound. The results are shown in Table 2.

TABLE 2

Endotoxin units 0.25 EU. 0.10 U.E. 0.05 U.E. sPG-3 (2.5 \ mug) without gel without gel without gel sPG-3 (1.0 \ mug) without gel without gel without gel sPG-3 (0.5 \ mug) ++ ++ without gel without protein added ++ ++ ++

These results show that since the gelation inhibition can be overcome by increasing the LPS concentration, the interaction with LPS is responsible for the absence of gelation, rather than interfering with the cascade of the gelation enzyme.

Example 5 Activity of linearized forms

nPG-1 and nPG-3 were converted to the linear form using a reducing agent to convert the disulfide bonds into sulfhydryl groups, which were then stabilized by alkylation with iodoacetamide.

The capacity of both PG-1 and Cyclic PG-3 as linearized to inhibit gelation in the standard LAL test was then evaluated as described in Example 4 and the results are shown in the Table 3.

TABLE 3

Peptide 5 \ mug 2.5 \ mug 1.0 \ mug 0.25 \ mug nPG-1 without gel without gel ++ ++ ++ cam -nPG-1 without gel without gel ++ ++ ++ nPG-3 without gel without gel ++ ++ ++ cam -nPG-3 without gel without gel ++ ++ ++

These results show that the forms linearized and cyclic protegrins are equally capable of inhibit gelation and bind endotoxins.

The antimicrobial activity of the linearized forms was also compared with that of the native protegrins. Both cyclic and linearized forms of the protegrins tested continue to show antimicrobial activity, although the efficacy of these peptides as antimicrobials depends on the nature of the target organism and the test conditions. The antimicrobial activity of native PG-1 and its linearized form ( cam -PG-1) and PG-3 and its linearized form ( cam -PG-3) were tested according to the procedure set forth in Example1 as described by Lehrer, RI and others J Immunol Meth (1991) 137 : 167-173. The results are shown in Figures 6a-6f.

Figures 6a and 6b show the antimicrobial activity of these peptides in the concentration range of 20 µg / ml-125 µg / ml with respect to E. coli ML-35P both in 10 mM phosphate-citrate buffer, pH 6.5 (Figure 6a) as in the presence of this buffer plus 100 mM NaCl (Figure 6b). Both protegrins showed a strong antimicrobial activity with respect to this organism; the linear form was slightly more potent in the presence of the buffer alone than what the cyclic form was; on the other hand, the cyclic form was more potent than the linear form under isotonic conditions.

Figures 6c and 6d show the antimicrobial effect with respect to L. monocytogenes . In Figure 6c where the buffer just mentioned above was used, both cyclic and linearized forms of the protegrins showed a strong antimicrobial activity and both were approximately equally effective over the concentration range tested (20 µg / ml-125 µg / ml)

Figure 6d shows the effect with respect to L. monocytogenes in the presence of this buffer plus 100 mM NaCl over the same concentration range. The cyclic form retained a strong antimicrobial activity with a slightly higher concentration dependence. Linearization seemed to reduce activity significantly although high concentrations were still able to show an antimicrobial effect.

The yeast C. albicans was tested with the results shown in Figures 6e and 6f. Figure 6e shows that all forms of these protegrins were antimicrobial in a dose-dependent manner over the previous concentration range when tested in the presence of only 10 mM phosphate buffer, although linearized peptides were slightly less effective. Figure 6f shows the results of the same test carried out in the presence of buffer plus 100 mM NaCl. While the cyclic forms retained approximately the same level of antimicrobial effect, the activity of the linearized forms decreased greatly so that at concentrations below 100 µg / ml of the protegrin, virtually no antimicrobial effect was observed. However, at higher concentrations of 130 µg / ml, a moderate antimicrobial effect was observed.

Thus, depending on the target microorganism and the conditions used, both cyclic and linearized forms of Protegrins have antimicrobial activity.

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Example 6 Antimicrobial activity under conditions suitable for eye treatment

Solutions for contact lenses are typically formulated with physiological saline buffered with borate and They may or may not contain EDTA. Amide-shaped protegrins Synthetic PG-3 and synthetic PG acid were generally tested in the test described in Example 1 in the that all coating gels contained 25 mM borate buffer, pH 7.4, 1% (v / v) triptocase soy broth (0.3 µg / ml powder TSB) and 1% agarose. Additions include both 100 mM NaCl, EDTA 1 mM or a combination thereof. Other test compounds used as witnesses were the NP-1 defensin and the lysozyme Response dose curves were determined.

Table 4 shows the concentrations estimated minimum bactericides in µg / ml of the various test compounds

TABLE 4

Estimated minimum fungicidal concentrations (\ mug / ml) Peptide tampon + EDTA + NaCl + EDTA \ textamp NaCl sPG-3 amide 13.0 9.5 4.1 3.1 sPG-3 acid 15.0 9.5 4.6 3.7 NP-1 35.0 45.0 > 200 > 200 lysozyme 75.0 45.0 > 200 > 200

Although protegrins are somewhat less active in 25 mM of borate buffered saline than in 25 mM phosphate buffer, the antimicrobial activity is enhanced by the addition of saline Physiologically and modestly enhanced by 1 mM EDTA, as shown In the table.

A similar test was carried out with Candida albicans as the target organism with the results shown in Table 5, which also show estimates of minimum fungicidal concentrations.

TABLE 5

Concentrations estimated minimum fungicides (\ mug / ml) Peptide borate buffer Tampon borate Borate buffer 25 mM + 120 mM NaCl + EDTA \ textamp NaCl nPG-3 32.0 9.0 8.0 sPG-3 amide 19.0 7.7 7.0 sPG-3 acid 19.0 9.2 9.3 NP-1 23.0 60.0 65.0 HNP-1 25.0 > 200 > 200

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Table 6 shows the results of similar experiments carried out with L. monocytogenes as the target.

TABLE 6

Concentrations estimated minimum bactericides (mug / ml) Peptide borate buffer Tampon borate Borate buffer 25 mM + 120 mM NaCl + EDTA \ textamp NaCl nPG-3 25.0 7.0 5.7 sPG-3 amide 21.0 5.7 5.2 sPG-3 acid 30.0 7.0 7.0 NP-1 20.0 11.0 3.8 HNP-1 11.0 > 200 > 200

The results shown indicate that these compounds are able to exert their antimicrobial effects in conditions typically associated with conditions suitable for eye care products.

Example 7 Recovery of cDNA clones and a new cDNA that encodes for a protegrin CDNA generation and PCR amplification

Total RNA was extracted from the marrow cells bone of a young red Duroc pig with guanidinium thiocyanate. A Total RNA was used to synthesize the first strand of cDNA, with 20 pmoles of Oligo primer (dT) and 200 U of reverse transcriptase of Moloney murine leukemia virus (M-MLV) (Clontech Laboratory, Palo Alto, CA) in a reaction volume total 20 \ mul. Two PCR primers were prepared. The primer homosense (5 '-GTCGGAATTCATGGAGACCCAGAG (A or G) GCCAG-3 ') corresponded to the 5' regions of cDNA of PG-2 and PR-39 and contained a site of EcoRI restriction. The antisense primer (5 '-GTCGTCTAGA (C or G) GTTTCACAAGAATTTATTT-3 ') was complementary to the 3 'ends of PG-2 and PR-39 cDNA immediately preceding their poly A tails and contained a site of XbaI restriction. PCR was carried out in a volume of 50 µl using 1/10 volume of the previous pig cDNA as a mold, 25 primer pmoles and 2.5 units of AmpliTaq DNA polymerase (Perkin Elmer-Cetus). The reaction was carried out. for 30 cycles, with 1 minute of denaturation (94ºC) and stages of hybridization (60 ° C) and an extension stage of 2 min (72 ° C) per cycle.

CDNA cloning and sequencing

The amplified cDNA was fractionated by preparative agarose electrophoresis and set with ethidium bromide. The main fragment was trimmed, digested with EcoR I and Xba I endonucleases (New England Biolabs, Beverly, MA), subcloned into an M13mp18 bacteriophage vector, and transformed into competent E. coli XL1-Blue MRF 'cells (Stratagene, La Jolla, CA). DNA sequencing was performed with a team (US Biochemical Corp., Cleveland, OH). Nucleotide and protein sequences were analyzed with PC-GENE (Intelligenetics, Palo Alto, CA).

Northern transfers

Ten cups of total RNA were denatured in 50% formamide, separated by electrophoresis through gels 1% agarose in 0.62 M formaldehyde, and transferred to the membranes GeneScreen Plus (Dupont, Boston, MA) by hair transfer. The membrane was introduced in an oven at 80 ° C for 2 h, and hybridized with a probe labeled with 32 P in a hybridization buffer fast (Amersham, Arlington Height, IL).

The results of sequencing various clones encoding the various protegrins are summarized in Figure 7. The cDNA sequences of the PG-1, PG-3 and PG-4 protegrins contain 691 bases as previously shown for PG-2 by Storici, P. et al. Biochem Biophys Res Comm (1993) 196 : 1363-1368. The cDNAs show an upstream sequence that codes for 110 amino acids that appear identical for all protegrins. Additional differences, which are quite slight in nature, are shown in Figure 7.

The analyzes showed the presence of the PG-4 protegrin that has a sequence of amino acids of Formula (1) in which A 10 is an amino acid small and A 11 is a hydrophobic amino acid as distinguished from the previously known protegrins in which these residues are basic. The amino acid sequence of PG-4 is by both RGGRLCYCRGWICFCVGRG, in which 1, 2, or 3 amino acids in the N-terminal end can be suppressed.

Additional clones were obtained by amplification of retrotranscribed porcine bone cell RNA using an upstream primer corresponding to the 5 'end of PG-2 and another peptide associated with catelin, PR39, (Agerbeth B et al. , Eur J Biochem (1991) 202 : 849-854; Storici, P. et al. Biochem Biophys Res Comm (1993) 186 : 1058-1065) and a downstream primer that joins the region immediately preceding the poly A region. The resulting PCR product of 0 , Approximately 7 kb was subcloned into M13mp18 and recombinant plates were chosen for purification and sequencing. In this way, the sequences for the precursors of PG-1, PG-3 and PG-4 were recovered. All these peptides are encoded by a nucleotide sequence encoding a precursor that contains an additional amino acid sequence upstream of A 1 of the compound of formula 1 (as shown for PG-4 in Figure 7).

Example 8 Recovery of genomic DNA encoding for PG-1, PG-3, and PG-5

Large molecular genomic DNA was purified to from white blood cells of pig blood with a DNA kit from QIAGEN blood (QIAGEN, Chatsworth, CA). To amplify the genes of protegrin (PG), a PCR was performed using genomic DNA as mold.

The homosense primer (5 ' -GTCGGAATTCATGGAGACCCAGAG (A or G) GCCAG-3 ') is corresponds to the 5 'regions of PG cDNA, from Example 7 and provided an EcoRI restriction site. The antisense primer (5 '-GTCGTCTAGA (C or G) GTTTCACAAGAATTTATTT-3 ') was complementary to the 3 'ends of PG cDNAs immediately preceding their poly (A) tails and providing an XbaI restriction site. The reaction was carried out in a total volume of 50 µl, which It contained 200 ng of purified pig genomic DNA, 25 pmoles of each primer, 1 µl of 10 mM dNTP, 5 µl of PCR buffer 10X (200 mM Tris-HCl, 100 mM (NH4) 2, 20 mM MgSO4, 1% Triton X-100, 0.1% BSA), and 2.5 cloned Pfu DNA polymerase units (Stratagene, La Jolla, AC). Thirty cycles were performed, each with 1 min of denaturation at 94 ° C, 1 min of primer hybridization at 55 ° C, 2 min extension of the primer at 72 ° C, and a final extension stage at 72 ° C for 10 min.

The amplified PCR product was digested with EcoRI and XbaI, was trimmed from the agarose gel, purified, and ligated into the vector pBluescript KS + (Stratagene, La Jolla, CA) that had been digested with EcoRI and XbaI and purified. Both strands of DNA are sequenced by the dideoxy method using the Sequenase kit version 2.0 (United States Biochemical, Cleveland, OH), primers universal pBluescript and specific oligomeric primers based in genomic PG and cDNA sequences. An analysis was performed. computer of DNA sequences using the program PC-Gene (Intelligenetics, Palo Alto, CA).

A PCR product of about 1.85 kb is confirmed as related to protegrins by hybridization with a protegrin specific oligonucleotide probe complementary to nucleotides 403-429 of the cDNA sequences of protegrin. The PCR product was then subcloned into a pBluescript vector, and recombinant plasmids They were subjected to DNA purification and sequencing. Sequences genes for three different protegrins were identified PG-1, PG-3 and PG-5. Nucleotide sequences and amino acid sequences deducted are shown in Figure 8.

The comparison of cDNA and protegrin genes revealed that the coding regions of the protegrin genes they consisted of four exons, interrupted by three introns (Figures 8 and 9). The first exon contained the 5 'non-coding region and the codons for the first 66 amino acids of the prepropeptide of protegrin, including a signal peptide of 29 residues and 37 first residues of the catelina. Exons II and III were relatively small, only 108 and 72 bp respectively, and together They contained the following 60 residues of catelin. The last two cathine residues were in Exon IV, and were followed by Protegrin sequences. The sequences of the cutting sites and exon-intron splicing are shown in Table 7, and they conform to the consensus rule: all introns end in a doublet AG, preceded by an extension rich in T / C of 8-12 bases, while all introns start with GT, predominantly followed by the sequence A / G A / G G.

TABLE 7 Exon-Intron structure of the gene PG-1

Exon Size Donor of the site of cut and Intron Size Site Acceptor cut and splice in 5' splice in 3 ' one ? + 198 AAGGCCgtgagtcg one 405 ttgaccagGACGAG two 108 AACGGGgtgaggct two 152 ccttccagCGGGTG 3 72 AATGAGgtgagtgg 3 596 ggtcacagGTTCAA 4 313

The highly conserved region of catelina that covers exons I-IV and exon IV contains the complete sequence of the mature protegrin peptide followed by a amidation consensus sequence, a 3 'untranslated region, and the putative polyadenylation site. The three introns oscillate in size from 152 to 596 bp. If the protegrin genes are representative of other genes similar to catelin, the third intron of the peptides associated with catelin will be found that separates all except the last two wastes from the region of Highly preserved cathine of antimicrobial peptides variables encoded in exon IV. Such provision would favor recombination mechanisms that involve the association of various exons IV with the first three exons specifying the Preproregions containing catelina.

The family of protegrins present in a way natural thus contains at least 5 members. Figure 10 shows a comparison of the amino acid sequences of the five protegrins found so far in leukocytes of porcine. There is a complete homology in the positions 1-3, 5-9, 13 and 15-16.

The homology search of the genes of protegrin against EMBL / GenBank did not identify genes significantly homologous More specifically, the structures gene and nucleotide sequences of protegrins were very different from those of the defensins, which contain three exons in myeloid defensin genes, and two exons in the genes of the enteric defensin As expected, the search produced the great cDNA family corresponding to peptides associated with bovine, porcine and rabbit leukocyte catelins.

To further evaluate the genes related to protegrin, we explore a genomic library of porcine of approximately 2.3 x 10 5 clones in EMBL-3 SP6 / T7 with the protegrin cDNA labeled with 32 P, and 45 hybrid clones identified.

A genomic library of swine liver in phage EMBL3 SP6 / T7 was purchased from Clontech (Palo Alto, CA). E. coli strain K803 was used as a host, and phage plate DNA was transferred to nylon membranes (DuPont, Boston, MA). The filters were hybridized with pig PG-3 cDNA 691 labeled with 32 P. The filters were washed several times, finally at 60 ° C in 0.1x SSC and 0.1% SDS, and were exposed to X-ray film with an intensifying screen at -70 ° C. Positive clones were subjected to two additional rounds of low density plaque purification.

The purified DNA of the hybridized clones is digested with various restriction endonucleases (New England Biolabs, Beverly, MA), fractionated into 0.8% agarose gels, and transferred to a GeneScreen Plus membrane (DuPont, Boston, MA). The hybridization probes were labeled with 32 P and included cDNA of Porcine PG-3, oligonucleotide specific for protegrin marked 5'- complementary to nts 403-429 cDNA of PG-1, 2 and 3. For the cDNA probe, hybridization and washing conditions are They performed as for screening the library. For the probe oligonucleotide, the membranes were washed at 42 ° C in 0.1x SSC, SDS 0.1%

Southern blot analysis is carried out with purified DNA from positive clones by hybridization with protegrin cDNA and an oligonucleotide specific for protegrin complementary to nts 403-429 of the protegrin cDNA sequences. Although all clones hybridized with the complete cDNA probe, only about half of them hybridized with the probe specific for protegrin. A specific oligonucleotide probe for porcine profenin, another antimicrobial peptide derived from porcine leukocyte associated with catelin, hybridized with several of the clones that were not protegrins. These results confirm a) that the homologous proregion to preserved cateline is present within the same gene as mature antimicrobial peptides and not added for posttranscriptional events, and b) that the protegrins give account of about half of the genes related to the Catelina in pork.

A synthetic peptide corresponding to the PG-5 amino acid sequence was prepared and tested for antimicrobial activity against E. coli, L. monocytogenes and C. albicans . The results were compared with those obtained with a synthetically prepared PG-1. The results are shown in Figures 11a-11c. As shown in these graphical representations of the results, PG-5 has antimicrobial activity comparable to that of PG-1 against the three organisms tested.

Example 9 Preparation of Enantio PG-1

Using standard solid phase techniques, a protegrin was prepared having the amino acid sequence of PG-1, but in which each amino acid is in form D. This form of protegrin was tested against E. coli, L. monocytogenes, C albicans and other microbes in the absence and in the presence of proteases and otherwise as described for the agarose gel broadcasting test set forth in Example 1. The results are shown in Figures 12a-12g.

Figure 12a shows that both native PG-1 and PG-1 enantium in the absence of protease are equally effective in inhibiting the growth of E. coli . Figure 12b shows that neither trypsin nor chymotrypsin inhibit the antibacterial effect of the PG-1 enantium . Figure 12c shows that in the presence of these proteolytic enzymes, the ability of native PG-1 to inhibit the growth of L. monocytogenes is adversely affected, although, as shown in Figure 12d, in the absence of these proteases, PG-1 it is active comparable to a PG-1 enantium .

Example 10 Protegrin activity against STD pathogens

Table 8 summarizes protegrin activity PG-1 as compared to defensin HNP-1 against the growth of STD pathogens. In These results, `` active '' means that the peptide was effective at less than 10 µg / ml; moderately active indicates that it was active in 10-25 µg / ml; and slightly active means activity at 25-50 µg / ml. If no effect is obtained at 50-200 µg / ml the compound is considered inactive.

TABLE 8

Activity vs. STD human pathogens Protegrin PG-1 Defensin HNP-1 HIV-1 Active Slightly active Chlamydia trachomatis Active Slightly active  Treponema pallidum Active Inactive Neisseria gonorrhoeae Active Inactive Trichomonas vaginalis Moderately active Inactive Herpes simplex type 2 Moderately active Slightly active Herpes simplex type one Inactive Slightly active Hemophilus ducreyi Not tested Do not rehearsed papillomavirus human Not tested Not tested

Chlamydia trachomatis

Unlike other bacteria associated with STDs, Chlamydia requires an intracellular habitat for metabolic activity and binary fission. The life cycle is as follows: there is an extracellular form that is a metabolically inactive particle somewhat similar to a spore in its behavior, referred to as an elementary body (EC). The EC binds to the host cell and is ingested to form an internal vacuolar space often called an `` inclusion. '' The bacterium is reorganized into a delicate cross-linked body (CR) that is not infectious but that is metabolically active and that over a period of 48-72 hours undergoes a reform to the EC state. The ECs are then released from the cell. More than a layer of peptidoglycan, Chlamydia contains multiple disulfide bonds in cysteine-rich proteins for protection in the EC state.

The protegrins of the invention were tested for their antimicrobial activity against Chlamydia using the `` standard gold '' chlamydial culture system for clinical samples described by Clarke, LM in Clinical Microbiology Procedures Handbook II (1992), Isenberg, HT Ed. Am. Soc. Microbiol. Washington, DC; pp. 8.0.1 to 8.24.3.9, Briefly, McCoy cells (a mouse cell line) in EMEM with cycloheximide with fetal bovine serum (FBS) 10% are used as hosts. Prior to chlamydial inoculation, the maintenance medium is aspirated without altering the cell layer and the cell layer is maintained on a coverslip in a standard vial. Each vial is then inoculated with 100-300 µL of inoculum and centrifuged at 3500 xg for one hour at 20 ° C. The fluid is then aspirated and 1 ml of EMEM is added. The vials are capped and incubated at 37 ° C for 48 hours. After 48 hours the medium is aspirated again, the coverslips are rinsed twice with PBS and fixed with 300 µL of EtOH for 10 minutes. The EtOH is aspirated and the vials are allowed to dry; then a drop of PBS plus 30 µL of Syva Microtrak monoclonal antibody is added to the main protein of the Chlamydia outer membrane to stain it. After incubation at 37 ° C for 30 minutes, the cells are washed with distilled water and examined for inclusions that are easily recognizable as bright cytoplasmic vacuoles, with green apple staining. They represent the equivalent of a colony of bacteria released in standard bacterial culture media.

In the tests carried out below, C trachomatis serovar L2 (L2 / 434Bu) described by Kuo, CC and others in Nongynococcal Urethritis and Related Infections (1977), Taylor-Robinson, D. et al . Ed. Am. Soc was used Microbiol Washington, DC, pp. 322-326. The germ is prepared from a sonicated culture in L929 of mouse fibroblast cells, and partially purified by centrifugation. Since the host protein is still present in the aliquots of the germ, each batch of germ is assessed for concentration at the time of preparation with serial dilutions of ten times diluted to 2 x 10-9. The germ containing 9.2 x 10 6 UFI / ml is rapidly thawed at 37 ° C and diluted to 10 -2 with sucrose / phosphate / glycine salts to produce UFI of about 200 after preincubation at room temperature and to dilute the eukaryotic background protein.

In the initial tests, the peptides were prepared as standard solutions in glacial acetic acid 0.01%. 100 µL of the diluted chlamydial germ were aliquoted in tubes 1.5 ml eppendorf and 200 µL of the peptide was added tube antibiotic Aliquots of the standard peptide (and controls) they were incubated with the germ at room temperature for one hour, Two hours and four hours. For 10 minutes before the end of each incubation period, the maintenance means were aspirated to from the McCoy vials in preparation for inoculation and the standard crop The culture was then carried out in the presence and absence of peptides; in some cases, the peptides will added to the final concentration in the culture media together with preculture incubation. The trial was evaluated microscopically.

The results using 50 µg of protegrin per addition were dramatic. In control cultures, where no peptides were added, 222-460 inclusions were counted. In all protocols where protegrin was added, both before the Chlamydia germ was added to the cells, as before and after, no inclusions were found. Similar results were obtained with additions of 20 µg of taquiplesin. The NP-1 and HNP-1 defensins had minor protective effects. In summary, the protegrins tested show antimicrobial activity against Chlamydia .

In the following series of experiments, they used various concentrations of protegrin (1 µg, 12.5 \ mug, 25 \ mug and 50 \ mug) in the two hours of preincubation. Concentrations as low as 12.5 µg reduced the number of Zero inclusions Even at a concentration of 1 µg / ml, the number of inclusions dropped dramatically from around 110 to about 30.

In the following group of experiments, the effect of the presence of serum was tested. Chlamydia germ was pre-incubated for two hours with and without 10% FBS and also with or without protegrin at 25 µg. Protegrin was highly effective both with and without serum, while the human HNP-2 defensin, used as a control, was reasonably effective in the absence of serum but only slightly effective in its presence.

Experiments were repeated but added. 25 µg of protegrin one after start of cultivation chlamydial, that is, after centrifugation and mixing of the medium end and one hour within the beginning of the cultivation period of 48 hours. Protegrin reduced the number of inclusions in approximately 57% of untreated witnesses although the HNP-2 was completely ineffective. Finally the protegrin (at 25 µg) was added to the chlamydial germ and then the Mix was grown immediately. In this case, without preincubation and without the interval of one hour after infection, protegrin It was minimally effective with or without serum.

The effect of serum is particularly important since for a topical agent to be effective in fighting Chlamydia infection, it must act in the presence of serum.

In addition, there are several mouse-based models for Chlamydia infection that can be used to assess the efficacy of protegrins. These include those described by Patton, DL and others in Chlamydial Infections (1990) Bowie, WR and other Eds. Cambridge University Press NY pp. 223-231; Swenson, CE and others J. Infect. Dis. (1983) pp. 1101-1107, and Barron, AL et al. J. Infect. Dis. (1981) 143 : 63-66.

Neisseria gonorrhoeae

In more detail, the ability of protegrins to inhibit N. gonorrhoeae was tested by a modification of the method of Miyasaki et al. , Antimicrob Agent Chemother (1993) 37 : 2710-2715. Transparent variants if pilli of FA 19 and F 62 strains were propagated on GCB agar plates containing glucose and iron supplements overnight at 37 ° C at 3.8% V / V CO2. These strains were chosen for their adaptability to the assay.

What had grown overnight withdrew of the agar plate and suspended in GCB broth containing supplements and sodium bicarbonate and grew with stirring at 37 ° C until the middle of the logarithmic phase. The culture was diluted 1: 100 in GCB broth to give about 10 6 CFU / ml and were placed Serial solutions in GCB agar.

The peptides dissolve in 0.01% acetic acid v / v to give a standard solution of 1 mg / ml and diluted in series. Ten µl of each solution is added to polystyrene tubes sterile containing 90 µl of diluted bacteria and tubes stir at 37 ° C for 45 minutes. The contents are diluted in 1:10 series and placed on GCB agar plates that are incubated in a CO2 incubator. The UFCs are counted after 24 hours and Logarithmic bactericidal activity is calculated.

The native PG-1, PG-1 synthetic, PG-3 synthetic amide and synthetic PG-3 without amidation give everyone a reduction of more than 5 logarithmic units in CFU per ml in this test. The native PG-2 (containing 16 amino acids) gave a reduction of 2.6 times.

In addition, the PG-1 enanthium , the PG-1 unidisulfide (C 6 -C 15), and the PG-1 unisulfide (C 8 -C 13) gave a reduction of more than 5 times in logarithmic units in CFU / ml in this test.

Treponema pallidum

The bactericidal activity against this organism, which is the etiological agent of syphilis, was also tested. The peptides were evaluated in a series of concentrations from 1,758 µg to 56.25 µg in 90 µl of normal unheated rabbit serum. Serum served as a nutrient for spirochetes to allow survival during incubation as well as to provide a source of complement. Ten µl of a T. pallidum suspension containing about 5 x 10 7 organisms / µl was added to each tube and the mixtures with the appropriate peptides were incubated at 34 ° C in 95% N 2 and 5 % of CO2. At zero time, prior to incubation, 4 hours and 16 hours, 24 randomly selected organisms were examined for the presence or absence of mobility. 50% of the immobility end point (IE 50) was calculated to indicate the concentration necessary to immobilize 50% of the spirochetes. In the presence of PG-1, the IE_ {50} at 0 and 4 hours was 2,717 µg and <1, 758 µg, respectively. The IE_ {50} of Taquiplesin were 5,231? And 2,539? For 0 and 4 hours. This was in contrast to the HNP and NP preparations that showed a small immobilizing capacity.

Herpex Simplex Virus

Using viral patterns prepared in cells VERO, grown in a minimal essential medium (MEM) with serum 2% fetal calf, the effect of various peptides on HSV 1 strain MacIntyre, a group of ten isolated clinical HSV 1, HSV-2G, and a group of ten isolated clinical HSV 2, all sensitive to 3 µM acyclovir were tested. Two lines Fibroblast cells, human W138 and equine CCL57, were used as targets and tests were performed by direct viral neutralization and peptide addition was delayed.

In the direct neutralization format, the virus it was pre-incubated with the peptides for 90 minutes before being added to tissue culture monolayers. In the addition format of delayed peptide, the virus was added and left 50 min that was adsorb to the target cells, then the monolayers were washed and the Peptides were added for 90 min. Finally, the monolayer is washed to remove the peptide and the cells were fed with MEM peptide free and cultured until infected monolayers untreated exhibited a cytopathic effect (CPE) 4+ (at about 60 hours)

In both formats antiviral activity was observed, but it was more pronounced with the peptide addition mode delayed. In experiments performed with W138 and CCL57 cells in Direct neutralization format, the PG-1 prevented completely that the HSV-2G caused CPE to concentrations of 50 µg / ml and 25 µg / ml, but these concentrations did not provide protection against HSV-1, which produced 4+ of CPE.

In the delayed peptide addition format, the PG-1 completely prevented CPE by HSV-2G at 35 µg / ml and 50 µg / ml and also fully protected against the HSV-2 group clinical at both concentrations.

Thus, PG-1 protected cells humans and animals against strain infection HSV-2 laboratory and clinics, even when Peptides were added as late as 60 min after the virus was introduced into the cell culture.

Thichomonas vaginallis

The C1 strain of trichomonas vaginallis (ATCC 30001) was grown as described by Gorrell, TE et al. , Carlsberg Res Comm (1984) 49 : 259-268. In experiments performed on RPMI + 1% heat-activated fetal calf serum, within a few minutes after exposure to PG-1 50 µg / ml, T. vaginallis (hitherto vigorously mobile) became stationary. Immediately afterwards, the organisms became permeable to trypan blue, and, for the next 15-30 minutes, they were lysed. As expected, such organisms were unable to grow when they were introduced into their usual growth medium (Diamond's medium). Organisms exposed to 25 µg / ml PG-3 retained their mobility.

Initial studies with two strains of T. vaginallis highly resistant to clinically isolated metronidazole, MR and TV strains showed both to be susceptible to PG-1, including C 8 -C 13 and C 6 -C 15 uni -disulfides and enantioPG -1 at concentrations of 100 and 50 µg / ml.

Example 11 Antiretroviral activity

Both synthetic and native PG-1 and native PG-2 were tested for antiviral activity against HIV strains using the method described in Miles, SA and others , Blood (1991) 78 : 3200-3208. Briefly, the mononucleated cell fraction is recovered from leukopacs of normal American Red Cross donors using a Ficoll-hypaque density gradient. The mononucleated cells are resuspended at 1 x 10 6 cells per ml in RPMI 1640 medium with 20% fetal bovine serum, 1% penicillin / streptomycin with fungizone and 0.5% PHA and incubated for 24 hours at 37 ° C in 5% of CO2. The cells are centrifuged, washed and then expanded for 24 hours in a growth medium.

HIV_ {JR-CSF} and HIV_ {JR-FL} cloned not adapted to the laboratory, were electroporated in peripheral blood mononucleated cells Human prepared as described above. The concentrations were determined and in general the values were used multiplicity of infection (MOI) of about 4,000 units infectious per cell (corresponding to 25-40 picograms per ml of HIV p24 antigen in the supernatant).

In the trial, HIV patterns prepared as before they were diluted to the correct number of MOI and the PBMs were added to 24-well plates at a concentration of 2 x 10 6 per ml. One µl of total volume is added to each well. The peptide to be tested is added in a growth medium to achieve the desired final concentration Then the appropriate number of MOI To test viral growth, 200 µl of supernatant they are removed on days 3 and 7 and the p24 antigen concentration is determined using a commercial trial (Coulter Immunology, Hialeah, Florida). Witnesses include duplicate sources that contain cells alone, cells plus peptide at 5 µg / ml of cells with virus but not peptide and cells with virus in the presence of AZT to 10-5 M-10-8 M.

Using this essay, it was shown that both, Native and synthetic PG-1 completely inhibit HIV infection at concentrations between 1-5 \ mug / ml; the IC_90 was <5 µg / ml. Addition time of peptide was then varied. Pretreated cells for 2 hours prior to virus addition, at the time of virus addition, or 2 hours after infection they showed antiviral activity for the peptide However, if PG-1 was added 24 hours after infection, there was no antiviral activity.

In addition, PG-2 shows similar activity but at a level of approximately 5-times less. Alternative antibiotics such as human defensins and rabbit defensins lacked potent activity in this assay. The results were similar for both HIV JR-CSF and HIV JR-FL which are isolated not adapted to the laboratory (Koyanagi, YS et al. , Science (1987) 236 : 819-822).

Protegrins show similar activity with compared to other retroviruses.

Example 12 Preparation of modified protegrins: kite forms and bullet

The kite and bullet shapes of PG-1 in which all X are alanine are synthesized using conventional chemistry Fmoc.

The crude synthetic peptide was reduced by addition of dithiothreitol (DTT) equal in weight to the synthetic peptide that had been dissolved at 10 mg peptide / ml in a solution that contained 6 molar guanidinium HCl, 0.5 molar tris buffer, and EDTA 2 mM, pH 8.05 and incubated for two hours at 52 ° C under an atmosphere of nitrogen. The mixture was passed through a 0.45 µm filter, was acidified with 1/20 (v / v) glacial acetic acid and subjected to a conventional purification of RP-HPLC with a column C-18. PG-1 bullet and kite synthetically reduced, purified by HPLC concentrated partially by vacuum centrifugation in a speed vac and it allowed to fold for 24 hours at room temperature outdoors in 0.1 M Tris pH 7.7 at low concentration (0.1 mg peptide / ml) for minimize the formation of cystine disulfide bonds intercatenaries The mixture was then concentrated and acidified with HOAC at a final concentration of 5% and was subjected to purification by RP-HPLC.

The purity of the final products of PG-1 bullet and kite was verified by AU-PAGE, analytical HPLC, and mass spectrometry by FAB. The AU-PAGE showed a unique band for the final product in each case. The observed MH + mass values It was 2093 in both cases.

Example 13 Antimicrobial activity of the comet and bullet forms

The PG-1 comet and bullet compounds prepared in Example 12 were tested for their antimicrobial activity using the radial diffusion assay described in Example 1 as published by Lehrer, RI et al. , J Immunol Meth (1991) 137 : 167-173, except that the coating agars contained 10 mM sodium phosphate buffer with a final pH of 7.4. As described in Example 1, 0.3 mg / ml triptocase soy broth powder and 1% agarose were also used in the coating agar. In some cases 100 mM NaCl or RPMI plus normal human serum (NHS) 2.5% were added to the agar.

In a first group of determinations, the bullet and comet forms of PG-1 were tested for antimicrobial activity against L. monocytogenes, E. faecium (VR) or S. aureus under these three groups of conditions. Figure 13 shows the result.

As shown, the bullet and comet forms were approximately as effective against these three bacteria using standard test conditions. However, when 100 mM NaCl was added to the agar, the comet forms appeared slightly less active than the bullet forms that appeared to have slightly enhanced antimicrobial activity against all three strains except S. aureus under these conditions. Likewise, when RPMI plus 2.5% NHS were added, the bullet forms were again more effective than the comet forms. The activity of the comet form against E. faecium was significantly lower in these conditions.

As shown in Figure 14, these forms of PG-1 were also tested against E. coli, K. pneumoniae and P. aeruginosa . These three microorganisms were inhibited by both forms of comet and bullet in standard conditions. This antimicrobial activity was also maintained in 100 mM NaCl and RPMI plus NHS.

Example 14 Synthesis of the snake form of PG-1

The snake form of PG-1 in the that all X are alanine was performed using standard methods by Synpep Inc., Dublin, CA and the MH + value in mass spectrometry of FAB- was 2031.3 as expected. The snake form was purified to achieve homogeneity by RP-HPLC.

Example 15 PG-1 snake antimicrobial activity

The snake-shaped PG-1 was tested for the same six organisms and using the same conditions as set forth in Example 13 with respect to the bullet and comet forms of PG-1. The results are shown in Figures 15 and 16. In this case, the native form of PG-1 of two cystines (native) was used as a control. While the snake form shows somewhat higher activity with respect to L. monocytogenes, E. faecium , and S. aureus under standard conditions, it is remarkably less effective than the native form in the presence of both 100 mM NaCl and RPMI plus NHS. The same pattern is followed, as shown in Figure 16 when the test organisms are E. coli, K. pneumoniae , and P. aeruginosa .

Example 16 Minimum inhibitory concentrations of protegrins

The minimum inhibitory concentrations (MICs) of a variety of protegrins were determined against the following organisms: Methicillin- resistant Staphilococcus aureus (MRSA), Pseudomonas aeruginosa (Psa), Vancomycin- resistant Enterococcus fecium (VREF), Candida albicans (Candid) and Escherichia coli (E. Co), and are shown in Table 9.

TABLE 9 Peptides with 17-18 amino acids

Sequence MRSA Ps to VREF Candi d E. Co IB-324 RGGGLCYCRPRFCVCVGR-OH 17.7 3.51 IB-218 RGGGLCYCFPKFCVCVGR 3.48 1.2 15.96 Protegrins uni-disulfide IB-225 RGGGLCYARPRFAVCVGR IB-226 RGGGLCYTRPRFTVCVGR 8.7 0.07 1.53

Claims (16)

1. A compound purified and isolated or recombinantly produced of formula A_ {1} -A_ {2} -A_ {3} -A_ {4} -A_ {5} -C_ {6} -A_ {7} -C_ {8} -A_ {9} -A_ {10} - A_ {11} -A_ {12} - C_ {13} -A_ {14} -C_ {15} -A_ {16} - (A_ {17} -A_ {18}) \ eqnum {(1)} and the acylated and / or C-terminal acylated or esterified N-terminal forms thereof, which is either in the linear form optionally stabilized in the -SH or in a form joined by cystine bridges, wherein A 1 is a basic amino acid; each of A_ {2} and A_ {3} is independently a small amino acid; each of A_ {5}, A_ {7}, A_ {14} is independently a hydrophobic amino acid; A4 is a basic or small amino acid; each of A_ {9}, A_ {12}, and A_ {16} is independently a basic, hydrophobic, neutral / polar amino acid or small; A 10 is proline; A 11 is a basic, neutral / polar amino acid, hydrophobic or small or is proline; A_ {17} is not present or, if it is, it is a basic, neutral / polar, hydrophobic or small amino acid; A_ {18} is not present or, if it is, it is a basic, hydrophobic, neutral / polar or small amino acid, or a form modified from Formula (1) and its N-terminal forms acylated and / or C-terminal amidated or esterified, in which at least one of the 4 cysteines is replaced independently by a hydrophobic amino acid or an amino acid small; provided that the compound of Formula (1) must have a load of +3 or greater. 2. The compound of claim 1, which It contains two cystine bridges. 3. The compound of claim 1, which it contains a cystine bridge, which is C_ {6} -C_ {15} or C_ {8} -C_ {13}. 4. The compound of claim 1, which is linearly 5. The compound of any of the claims 1-4, wherein the carboxyl end C-terminal is of the formula selected from the group consisting of COOH or salts thereof; COOR, CONH_ {2}, CONHR, and CONR_ {2} in which each R is independently hydrocarbyl (1-6C); and / or in which the amino group at the end N-terminal is of the formula NH2 or NHCOR, in the that R is hydrocarbyl (1-6C); and / or in which each of A_ {1} and A_ {9} is independently selected from the group consisting of R, K and Har; and / or in which each of A_ {2} and A_ {3} is independently selected from the group consisting of G, A, S and T; and / or wherein A 4 is R or G; and / or in which each of A_ {5}, A_ {14} and A_ {16} is independently selected from the group consisting in I, V, Nle, L and F; and / or in which each of A_ {7} and A_ {12} is independently selected from the group consisting of I, V, L, W, Y and F; and / or in which A_ {11} is R or W. 6. A compound of claim 1, which is selected from the group consisting of PG-5 : RGGRLCYCRPRFCVCVGR; IB-324 : R-G-G-G-L-C-Y-C-R-P-R-F-C-V-C-V-G-R-OH; IB-218 : R-G-G-G-L-C-Y-C-F-P-K-F-C-V-C-V-G-R; IB-225 : R-G-G-G-L-C-Y-A-R-P-R-F-A-V-C-V-G-R; IB-226 : R-G-G-G-L-C-Y-T-R-P-R-F-T-V-C-V-G-R. and the amidated forms thereof both in linear form as in the form of cystine bridges. 7. The compound of any of the claims 1-6, wherein all amino acids are in the D configuration. 8. A recombinant expression system for the production of an antimicrobial peptide that has the sequence amino acid of the compound of any of the claims 1-6, expression system comprising a nucleotide sequence encoding said peptide operably linked to the control sequences to effect the expression. 9. A modified recombinant host cell to contain the expression system of claim 8. 10. A method to produce a peptide antimicrobial or antiviral or an intermediate peptide for this, method comprising culturing the modified host cells of claim 9, under conditions in which said peptide is produces; and recover the peptide from the culture. 11. The method of claim 10, which further comprising effecting cystine bonds of said peptide and / or modify the N-terminal end and / or the end C-terminal of said peptide. 12. A pharmaceutical composition for use antimicrobial or antiviral comprising the compound of any of claims 1-7 in admixture with at less a pharmaceutically acceptable excipient. 13. A composition for application to plants or plant environments to confer infection resistance microbial or viral in plants, which comprises the compound of any of claims 1-7 in admixture with at least one environmentally acceptable diluent. 14. A method to prevent the growth of a virus or microbe, method comprising contacting a composition that supports the growth of said virus or microbe with an amount of the compound of any of the claims 1-7 effective to prevent such growth. 15. A method to deactivate endotoxin from gram-negative bacteria, a method that includes contacting said endotoxin with an amount of the compound of any of claims 1-7 effective for deactivate said endotoxin. 16. Antibodies specifically reactive with the compound of any of the claims 1-7.